scholarly journals Use of SAM2® Biotin Capture Membrane in Microarrayed Compound Screening (μARCS) Format for Nucleic Acid Polymerization Assays

2003 ◽  
Vol 8 (3) ◽  
pp. 273-282 ◽  
Author(s):  
Xiaoling Xuei ◽  
Caroline A. David ◽  
Tim R. Middleton ◽  
Ben Lim ◽  
Ron Pithawalla ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
High Throughput Screening ◽  
Dna Helicase ◽  
Polymerization Reaction ◽  
Technology Journal ◽  
White Spots ◽  
Compound Screening ◽  
High Salt Buffer ◽  
Potential Inhibitors ◽  
Acid Substrate

Microarrayed compound screening format (μARCS) is a novel high-throughput screening technology that uses agarose matrices to integrate various biochemical or biological reagents in the assay. To evaluate the feasibility of using the μARCS technology for nucleic acid polymerization assays, the authors developed HIV reverse transcription (RT) and E1-dependent human papillomavirus (HPV) replication assays in this format. HIV RT is an RNA-dependent DNA polymerase, whereas HPV E1 is a DNA helicase. To ensure the efficient capture of the nucleic acid polymerization reaction and to minimize the nonspecific binding, the authors used a SAM2® biotin capture membrane in the assay. In both studies, the nucleic acid substrate was biotinylated on one end and was bound to the SAM2® membrane. A low melting-point agarose gel containing the rest of the reaction components was first placed on a polystyrene sheet spotted with compounds to allow passive diffusion of the compounds into the gel. The gel was removed from the compound sheet and applied to the SAM2® membrane with the immobilized nucleic acid template to initiate the polymerization. After the incubation, the membrane was washed with a high-salt buffer and exposed for imaging. Potential inhibitors can be seen as white spots on a dark background. The sensitivity for the known inhibitors appears to be comparable in μARCS as in a traditional 96-well plate assay. The methodology described in this paper further expands the applications of μARCS technology. ( Journal of Biomolecular Screening 2003:273-282)

scholarly journals Novel high-throughput screen identifies an HIV-1 reverse transcriptase inhibitor with a unique mechanism of action

2014 ◽  
Vol 462 (3) ◽  
pp. 425-432 ◽  
Author(s):  
Chih-Wei Sheen ◽  
Onur Alptürk ◽  
Nicolas Sluis-Cremer
Keyword(s):  
Nucleic Acid ◽  
High Throughput ◽  
High Throughput Screening ◽  
Transcriptase Inhibitor ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Reverse Transcriptase Inhibitor ◽  
Acid Substrate ◽  
Hiv 1 ◽  
Unique Mechanism

We have developed a FRET-based high-throughput screening assay for the AZT-MP excision activity of HIV-1 RT. Using this assay we screened 7265 compounds and identified APEX57219 {3,3′-[(3-carboxy-4-oxo-2,5-cyclohexadien-1-lidene)methylene]bis[6-hydroxy-benzoic acid]}. APEX75219 inhibits the interaction between RT and the nucleic acid substrate.

scholarly journals Application of Micro Arrayed Compound Screening (pcARCS) to Identify Inhibitors of Caspase-3

2002 ◽  
Vol 7 (4) ◽  
pp. 317-323
Author(s):  
Sujatha M. Gopalakrishnan ◽  
Jarkko Karvinen ◽  
James L. Kofron ◽  
David J. Burns ◽  
Usha Warrior
Keyword(s):  
High Throughput Screening ◽  
Caspase 3 ◽  
Imaging System ◽  
Time Resolved ◽  
Charge Coupled Device ◽  
Assay Format ◽  
Compound Screening ◽  
Screening Platform ◽  
Caspase Substrate ◽  
Potential Inhibitors

Micro Arrayed Compound Screening (pARCS) is a miniaturized ultra-high-throughput screening platform developed at Abbott Laboratories. In this format, 8640 discrete compounds are spotted and dried onto a polystyrene sheet, which has the same footprint as a 96-well plate. A homogeneous time-resolved fluorescence assay format (LANCE) was applied to identify the inhibitors of caspase-3 using a peptide substrate labeled with a fluorescent europium chelate and a dabcyl quencher. The caspase-3 enzyme was cast into a thin agarose gel, which was placed on a sheet containing test compounds. A second gel containing caspase substrate was then laid above the enzyme gel to initiate the reaction. Caspase-3 cleaves the substrate and separates the europium from the quencher, giving rise to a time-resolved fluorescent signal, which was detected using a ViewLux charge-coupled device imaging system. Potential inhibitors of caspase-3 appeared as dark spots on a bright fluorescent background. Results from the pARCS assay format were compared to those from a conventional 96-well plate-screening format.

High-Throughput Screening to Identify Small Molecules That Selectively Inhibit APOL1 Protein Level in Podocytes

2021 ◽  
pp. 247255522110262
Author(s):  
Jonathan Choy ◽  
Yanqing Kan ◽  
Steve Cifelli ◽  
Josephine Johnson ◽  
Michelle Chen ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Strategy ◽  
Time Resolved ◽  
Protein Levels ◽  
Increased Risk ◽  
Compound Screening ◽  
High Throughput Screens ◽  
Screening Library

High-throughput phenotypic screening is a key driver for the identification of novel chemical matter in drug discovery for challenging targets, especially for those with an unclear mechanism of pathology. For toxic or gain-of-function proteins, small-molecule suppressors are a targeting/therapeutic strategy that has been successfully applied. As with other high-throughput screens, the screening strategy and proper assays are critical for successfully identifying selective suppressors of the target of interest. We executed a small-molecule suppressor screen to identify compounds that specifically reduce apolipoprotein L1 (APOL1) protein levels, a genetically validated target associated with increased risk of chronic kidney disease. To enable this study, we developed homogeneous time-resolved fluorescence (HTRF) assays to measure intracellular APOL1 and apolipoprotein L2 (APOL2) protein levels and miniaturized them to 1536-well format. The APOL1 HTRF assay served as the primary assay, and the APOL2 and a commercially available p53 HTRF assay were applied as counterscreens. Cell viability was also measured with CellTiter-Glo to assess the cytotoxicity of compounds. From a 310,000-compound screening library, we identified 1490 confirmed primary hits with 12 different profiles. One hundred fifty-three hits selectively reduced APOL1 in 786-O, a renal cell adenocarcinoma cell line. Thirty-one of these selective suppressors also reduced APOL1 levels in conditionally immortalized human podocytes. The activity and specificity of seven resynthesized compounds were validated in both 786-O and podocytes.

scholarly journals Stress-Based High-Throughput Screening Assays to Identify Inhibitors of Cell Envelope Biogenesis

Antibiotics ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 808
Author(s):  
Maurice Steenhuis ◽  
Corinne M. ten Hagen-Jongman ◽  
Peter van Ulsen ◽  
Joen Luirink
Keyword(s):  
Outer Membrane ◽  
High Throughput ◽  
Stress Responses ◽  
High Throughput Screening ◽  
Structural Integrity ◽  
Cell Envelope ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Compound Screening ◽  
Outer Membrane Biogenesis

The structural integrity of the Gram-negative cell envelope is guarded by several stress responses, such as the σE, Cpx and Rcs systems. Here, we report on assays that monitor these responses in E. coli upon addition of antibacterial compounds. Interestingly, compromised peptidoglycan synthesis, outer membrane biogenesis and LPS integrity predominantly activated the Rcs response, which we developed into a robust HTS (high-throughput screening) assay that is suited for phenotypic compound screening. Furthermore, by interrogating all three cell envelope stress reporters, and a reporter for the cytosolic heat-shock response as control, we found that inhibitors of specific envelope targets induce stress reporter profiles that are distinct in quality, amplitude and kinetics. Finally, we show that by using a host strain with a more permeable outer membrane, large-scaffold antibiotics can also be identified by the reporter assays. Together, the data suggest that stress profiling is a useful first filter for HTS aimed at inhibitors of cell envelope processes.

scholarly journals Innovative Therapeutic Approaches for Huntington’s Disease: From Nucleic Acids to GPCR-Targeting Small Molecules

2021 ◽  
Vol 15 ◽  
Author(s):  
Hidetoshi Komatsu
Keyword(s):  
Nucleic Acid ◽  
Huntington's Disease ◽  
Huntington’S Disease ◽  
High Throughput Screening ◽  
Neurodegenerative Disorder ◽  
Direct Injection ◽  
Cag Repeat ◽  
Great Promise ◽  
Nucleic Acid Delivery ◽  
Small Interfering Rnas

Huntington’s disease (HD) is a fatal neurodegenerative disorder due to an extraordinarily expanded CAG repeat in the huntingtin gene that confers a gain-of-toxic function in the mutant protein. There is currently no effective cure that attenuates progression and severity of the disease. Since HD is an inherited monogenic disorder, lowering the mutant huntingtin (mHTT) represents a promising therapeutic strategy. Huntingtin lowering strategies mostly focus on nucleic acid approaches, such as small interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs). While these approaches seem to be effective, the drug delivery to the brain poses a great challenge and requires direct injection into the central nervous system (CNS) that results in substantial burden for patients. This review discusses the topics on Huntingtin lowering strategies with clinical trials in patients already underway and introduce an innovative approach that has the potential to deter the disease progression through the inhibition of GPR52, a striatal-enriched class A orphan G protein-coupled receptor (GPCR) that represents a promising therapeutic target for psychiatric disorders. Chemically simple, potent, and selective GPR52 antagonists have been discovered through high-throughput screening and subsequent structure-activity relationship studies. These small molecule antagonists not only diminish both soluble and aggregated mHTT in the striatum, but also ameliorate HD-like defects in HD mice. This therapeutic approach offers great promise as a novel strategy for HD therapy, while nucleic acid delivery still faces considerable challenges.

scholarly journals Skipping and sliding to optimize target search on protein-bound DNA and RNA

2020 ◽  
Author(s):  
Misha Klein ◽  
Tao Ju Cui ◽  
Ian MacRae ◽  
Chirlmin Joo ◽  
Martin Depken
Keyword(s):  
Nucleic Acid ◽  
Single Molecule ◽  
Diffusion Limit ◽  
Optimal Search ◽  
Dna And Rna ◽  
Argonaute Proteins ◽  
Large Pool ◽  
Search Speed ◽  
Crowded Environments ◽  
Acid Substrate

Rapidly finding a specific nucleic-acid sequences in a large pool of competing off-targets is a fundamental challenge overcome by all living systems. To optimize the search and beat the diffusion limit, it is known that searchers should spend time sliding along the nucleic-acid substrate. Still, such sliding generally has to contend with high levels of molecular crowding on the substrate, and it remains unclear what effect this has on optimal search strategies. Using mechanistic modelling informed by single-molecule data, we show how sliding combined with correlated short-ranged skips allow searchers to maintain search speed on densely crowded substrates. We determine the conditions of optimal search, which show that an optimized searchers always spend more than half its time skipping and sliding along the substrate. Applying our theory to single-molecule data, we determine that both human and bacterial Argonaute proteins alternate between sliding 10 nt and skipping 30 nt along the substrate. We show that this combination of skipping and sliding lengths allows the searcher to maintain search speeds largely unaffected by molecular roadblocks covering up to 70% of the substrate. Our novel combination of experimental and theoretical approach could also help elucidate how other systems ensure rapid search in crowded environments.

scholarly journals A Phenylfurocoumarin Derivative Reverses ABCG2-Mediated Multidrug Resistance In Vitro and In Vivo

2021 ◽  
Vol 22 (22) ◽  
pp. 12502
Author(s):  
Shoji Kokubo ◽  
Shinobu Ohnuma ◽  
Megumi Murakami ◽  
Haruhisa Kikuchi ◽  
Shota Funayama ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Cancer Cells ◽  
High Throughput Screening ◽  
Atp Hydrolysis ◽  
Pheophorbide A ◽  
Chemical Library ◽  
Hct 116 ◽  
Potential Inhibitors

The ATP-binding cassette subfamily G member 2 (ABCG2) transporter is involved in the development of multidrug resistance in cancer patients. Many inhibitors of ABCG2 have been reported to enhance the chemosensitivity of cancer cells. However, none of these inhibitors are being used clinically. The aim of this study was to identify novel ABCG2 inhibitors by high-throughput screening of a chemical library. Among the 5812 compounds in the library, 23 compounds were selected in the first screening, using a fluorescent plate reader-based pheophorbide a (PhA) efflux assay. Thereafter, to validate these compounds, a flow cytometry-based PhA efflux assay was performed and 16 compounds were identified as potential inhibitors. A cytotoxic assay was then performed to assess the effect these 16 compounds had on ABCG2-mediated chemosensitivity. We found that the phenylfurocoumarin derivative (R)-9-(3,4-dimethoxyphenyl)-4-((3,3-dimethyloxiran-2-yl)methoxy)-7H-furo [3,2-g]chromen-7-one (PFC) significantly decreased the IC50 of SN-38 in HCT-116/BCRP colon cancer cells. In addition, PFC stimulated ABCG2-mediated ATP hydrolysis, suggesting that this compound interacts with the substrate-binding site of ABCG2. Furthermore, PFC reversed the resistance to irinotecan without causing toxicity in the ABCG2-overexpressing HCT-116/BCRP cell xenograft mouse model. In conclusion, PFC is a novel inhibitor of ABCG2 and has promise as a therapeutic to overcome ABCG2-mediated MDR, to improve the efficiency of cancer chemotherapy.

scholarly journals Head and Neck Cancer Stem Cell-Enriched Spheroid Model for Anticancer Compound Screening

Cells ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1707
Author(s):  
Larisa Goričan ◽  
Boris Gole ◽  
Uroš Potočnik
Keyword(s):  
Head And Neck Cancer ◽  
Stem Cell ◽  
Cancer Stem Cell ◽  
Head And Neck ◽  
Neck Cancer ◽  
High Throughput Screening ◽  
Proliferating Cells ◽  
Detection Systems ◽  
Compound Screening ◽  
Fadu Cells

Cancer stem cells (CSCs), a rare cell population in tumors, are resistant to conventional chemotherapy and thus responsible for tumor recurrence. To screen for active compounds targeting CSCs, a good CSC-enriched model compatible with high-throughput screening (HTS) is needed. Here, we describe a new head and neck cancer stem cell-enriched spheroid model (SCESM) suitable for HTS analyses of anti-CSC compounds. We used FaDu cells, round-bottom ultra-low adherent (ULA) microplates, and stem medium. The formed spheroids displayed increased expression of all stem markers tested (qRT-PCR and protein analysis) in comparison to the FaDu cells grown in a standard adherent culture or in a well-known HTS-compatible multi-cellular tumor spheroid model (MCTS). Consistent with increased stemness of the cells in the spheroid, confocal microscopy detected fast proliferating cells only at the outer rim of the SCESM spheroids, with poorly/non-proliferating cells deeper in. To confirm the sensitivity of our model, we used ATRA treatment, which strongly reduced the expression of selected stem markers. Altogether, we developed a CSC-enriched spheroid model with a simple protocol, a microplate format compatible with multimodal detection systems, and a high detection signal, making it suitable for anti-CSC compounds’ HTS.

scholarly journals Epigenetic Compound Screening Uncovers Small Molecules for Reactivation of Latent HIV-1

2020 ◽  
Vol 65 (1) ◽  
pp. e01815-20
Author(s):  
Ariane Zutz ◽  
Lin Chen ◽  
Franziska Sippl ◽  
Andreas Humpe ◽  
Christian Schölz
Keyword(s):  
High Throughput Screening ◽  
Mononuclear Cells ◽  
Model Systems ◽  
Reporter System ◽  
Peripheral Blood Mononuclear ◽  
Compound Screening ◽  
Immunological Destruction ◽  
New Treatment ◽  
Latent Hiv ◽  
Hiv 1

ABSTRACTDuring infection with the human immunodeficiency virus type 1 (HIV-1), latent reservoirs are established that circumvent full eradication of the virus by antiretroviral therapy (ART) and are the source for viral rebound after cessation of therapy. As these reservoirs are phenotypically indistinguishable from infected cells, current strategies aim to reactivate these reservoirs, followed by pharmaceutical and immunological destruction of the cells. Here, we employed a simple and convenient cell-based reporter system, which enables sample handling under biosafety level (BSL)-1 conditions, to screen for compounds that were able to reactivate latent HIV-1. The assay showed a high dynamic signal range and reproducibility with an average Z-factor of 0.77, classifying the system as robust. The assay was used for high-throughput screening (HTS) of an epigenetic compound library in combination with titration and cell-toxicity studies and revealed several potential new latency-reversing agents (LRAs). Further validation in well-known latency model systems verified earlier studies and identified two novel compounds with very high reactivation efficiencies and low toxicity. Both drugs, namely, N-hydroxy-4-(2-[(2-hydroxyethyl)(phenyl)amino]-2-oxoethyl)benzamide (HPOB) and 2′,3′-difluoro-[1,1′-biphenyl]-4-carboxylic acid, 2-butylhydrazide (SR-4370), showed comparable performances to other already known LRAs, did not activate CD4+ T cells, and did not cause changes in the composition of peripheral blood mononuclear cells (PBMCs), as shown by flow cytometry analyses. Both compounds may represent effective new treatment possibilities for reversal of latency in HIV-1-infected individuals.

Defining the nucleic acid substrate for somatic hypermutation

1992 ◽  
Vol 70 (2) ◽  
pp. 129-144 ◽  
Author(s):  
EJ Steele ◽  
HS Rothenfluh ◽  
GW Both
Keyword(s):  
Nucleic Acid ◽  
Somatic Hypermutation ◽  
Acid Substrate
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